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CHAPTE CHAPTER R1 ERYTHROCYTE ERYTHROCYTE FRAGILITY TEST
Aim The aim of this chapter is to study the effect of solutions with different osmolarities on the integrity of erythrocytes. This is one of the clinical tests used to examine the fragility of erythrocytes.
Introduction When an erythrocyte is placed in a hypotonic s odium chloride -(NaCl) solution, a net influx of solvent (water) into the cell -will occur and the cell will swell. If the cell size reaches a certain point, the cell membrane will become leaky and haemoglobin will diffuse out (haemolysis). If the NaCl solution is hypotonic enough, the cell will rupture. The degree of haemolysis can be measured by determining the absorbance of the supernatant using a spectrophotometer. The degree of haemolysis in distilled water is considered maximum (100%), and the absorbance of this supernatant is taken as "100% haemolysis". In the erythrocyte fragility test, the erythrocyte is placed in NaCl solutions of decreasing osmolarity (i.e. 0.85%, 0.85%, 0.75% 0.75% ). The amou nt of haemol ysis that occurs in each solution can be deter mined, and a gra ph of " % ha emolysis vs NaCl concentration" can be plotted. In an abnormal erythrocyte that is more fragile than normal, haemolysis will
occur at a higher NaCl concentration (i.e. (i.e. 'earlier') than normal and the graph will shift to the right. In this experiment, we will also examine the fragility of erythrocytes (haemolysis) in several other solutions to understand the principles of diffusion of particles across the cell membrane.
Procedure 1. 2. 3.
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This experiment can can be conducted in in groups of 4 students. Label the centrifuge tubes as follows: 0.85%, 0.85%, 0.75%, 0.65%, 0.65%, 0.55%, 0.45%, 0.40%, 0.35%, 0.20%, 0.10% and 0%. Pipette 5 ml of each of the NaCl solution provide d into the appropriately labelled tubes. In the tube labelled 0%, pipette in 5 ml of distilled water. Using a micr opip ette , ad d 0.02 0.02 ml (20ul) of blood (containing an anticoagulant agent) into each tube. Cover the tube with parafilm, mix the conte nts of each tube gently and then allow the solution to settle for about 20 minutes at room temperature. Mix the conten ts in each tube again. Then, centrifuge the tubes at a speed of 2000 2000 rotations per mi nute for 5 minutes. Carefully transfer the super nata nts into clean and appropriately labelled cuvettes. Using a spectrophotometer, measure the absorbance of each supernatant, at a wavelength of 540 nm. For this procedure, use distilled water as a 'blank' (zero reading in the meter). Then obtain the absorbance reading of each supernatant. Compare each of these readings with that obtained from the 0% NaCl tube (distilled water = 100% haemolysis). Calculate the % haemolysi s for each supe rna tan t as follows:
10. Plot a gra ph of % haemolysis vs NaCl concentra tion. This represents the osmotic fragility curve for the blood sample.
11. Repeat Repeat the above procedures for each of the following following solutions: a. 150 150 mmo l/L urea b. 300 mm ol / L urea c. 300 mmo l/L urea +150 mmo l/L NaCl d. 300 mm ol / L glucose
